Transcript No Slide Title

BioSci 145A Lecture 11 - 2/12/2002
Transgenic technology and its implications
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Topics we will cover today
– how to get the DNA into cultured cells.
• chemical transfection of cultured cells
• cell fusion (won’t discuss)
• liposome-mediated transfer
• cationic dendrimers
• virus mediated
• electroporation
– how to get DNA into embryos
• biolistic gene transfer
• microinjection
• electrotransformation
• viral infection
– transgenic technology
• standard transgenesis
• gene targeting
– Dr. La Morte will discuss single cell microinjection
techniques
BioSci 145A lecture 11
page 1
©copyright
Bruce Blumberg 2000. All rights reserved
How to get DNA into cells - introduction
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General terminology
– transformation refers to the uptake of foreign DNA,
e.g. plasmid transformation of bacteria.
– Transfection, strictly speaking, refers to the transfer
of viral DNA
– when referring to animal cells, we tend to use the
term transfection to distinguish the transfer of DNA
from the “transformation” of a cancer cell.
Transfection efficiency varies greatly from one type of
cell line to another using any method.
– Must usually test several methods to determine
which one works best for your cells and hands.
Stable vs. transient transfections is also relevant.
– Supercoiled plasmids are best for transient
transfections, linear best for stable transfections
– stable transfectants usually have single integration
site with multiple copies integrated
– transient transfectants may replicate
extrachromosomally.
Observation is that cells that take up any DNA take up
all DNA
– e.g. if cells take up one type of plasmid from the
surroundings, they will take up all types
– enables co-transfection, introduction of multiple
plasmids/cell
– this is a fundamental and indispensable tool
BioSci 145A lecture 11
page 2
©copyright
Bruce Blumberg 2000. All rights reserved
How to get DNA into cells - introduction (contd)
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Many claims of superior transfection efficiency are made
by companies who sell reagents for transfection
– Caveat emptor, one San Diego company uses a
competitor’s product in house instead of the reagent
they promote.
– one of the largest profit margin items in the industry
• unless you own stock in a company selling the
reagents, make your own whenever possible
BioSci 145A lecture 11
page 3
©copyright
Bruce Blumberg 2000. All rights reserved
Chemical transfection - Ca3(PO4)2
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W. Szybalski (a very famous microbiologist) decided to
set up a system whereby mammalian cells could be
induced to take up DNA, much like bacteria - first
successful report in 1962.
– To maximize success he also developed the HAT
selection method.
– By analogy to bacterial transformation, it was
discovered that successful DNA transfer was
dependent on the formation of a co-precipitate of
DNA with calcium phosphate
• after the method was well understood in 1973, it
became widely used
• Graham and van der Eb (1973) Virology 52,
456-467 is the classic reference.
• Chen and Okayama (1987) Mol Cell Biol 7,
2745-52 (very high efficiency variant)
General principle is to form a precipitate of DNA that can
be taken up by endocytosis
– Mix DNA, in phosphate buffer with CaCl2 at precise
pH and an insoluble CaPO4 precipitate forms
• precision in pH is critical, alterations of as little
as 0.01 pH units affect efficiency
• leave on cells several hours to overnight
• wash ppt off and add fresh medium
– OR add DNA and buffer to cells at low (3%) CO2.
• Ppt forms automatically over time
BioSci 145A lecture 11
page 4
©copyright
Bruce Blumberg 2000. All rights reserved
Chemical transfection - Ca3(PO4)2 (contd.)
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advantages
– very simple
– very inexpensive
– extensive literature
– works for most cell types
disadvantages
– adherent cells only
– some touch and experience required to get good
precipitates
– not particularly efficient in many cell types
– many cells do not like adherent precipitate
– difficult to automate or perform as a high throughput
method
BioSci 145A lecture 11
page 5
©copyright
Bruce Blumberg 2000. All rights reserved
Chemical transfection - DEAE dextran
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diethylaminoethyl (DEAE) modified dextran is a
positively charged polymer
– many other charged polymers have been used with
varying degrees of success and reproducibility
• PEI - polyethyleneimine
• poly-L-lysine
• roll your own
DNA adheres to the polymer and remains soluble
by some unknown means, the complex interacts with the
cells and is taken up by endocytosis
advantages
– may work in cells that are refractory to other
methods
– gentle, not very toxic to cells
– works for cells in suspension
disadvantages
– doesn’t work well in many cell types
– doesn’t work well for stable transfectants
– unclear mechanism of action makes optimization
troublesome
– moderately expensive
– low throughput
BioSci 145A lecture 11
page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Lipofection - liposome mediated transfection
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produce unilamellar liposomes and allow DNA to interact
with them. Liposomes can be produced by:
– sonication
– extrusion through a small pore membrane
– dilution into aqueous medium
mix with cells and allow to interact
for a long time it was assumed that liposomes mediate fusion
with cell membranes. However endocytosis is now known to
be the mechanism
various formulations
– cationic lipids only, e.g. DOTAP
– mixture of cationic and neutral lipids, e.g. lipofectin
(DOTMA:DOPE)
– phospholipids
– cholesterol-related lipids
all work to some degree
advantages
– very simple to perform and optimize - anyone can do it.
– easy to automate, high throughput
– reliable and reproducible
– stable and transient assays work well
– works well with many cell types and in vivo
• adherent and nonadherent
BioSci 145A lecture 11
page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Lipofection - liposome mediated transfection (contd)
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disadvantages
– many formulations require use of serum free, or
serum reduced medium for good efficiency
• all types that use neutral lipids
– some formulations are unstable to oxygen
• DOTAP and other unsaturated lipids
– variable toxicity necessitates careful optimization for
many types (e.g. Lipofectin)
– VERY expensive to buy (but almost free to make)
– for example
• BMB-Roche sells 2 mg of DOTAP transfection
reagent for $285. This is enough for ~6 96-well
plates ($48/plate)
– 1 gram = $142,500
• pure DOTAP costs ~$400/gram from Avanti
Polar Lipids. Time and material to make
liposomes in vials about doubles this cost. About
$0.20/96-well plate
– Manufacturers lie quite a bit about the performance
of their reagents due to the profit margins
• many do not work well, others not at all
BioSci 145A lecture 11
page 8
©copyright
Bruce Blumberg 2000. All rights reserved
Cationic dendrimer mediated transfection
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polycationic polymers of various densities and patterns
(e.g. Superfect)
interact with DNA to form complexes
these interact with cells and are taken up by endocytosis
advantages
– may be more efficient than liposomes
– stable and easy to use
– low toxicity
– automation friendly, high throughput
– suspension or adherent cells
disadvantages
– expensive
– not readily possible to synthesize
BioSci 145A lecture 11
page 9
©copyright
Bruce Blumberg 2000. All rights reserved
Electroporation - electricity driven transfection
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principle is that brief, strong electrical pulse creates
transient pores in the cell membrane that allows exchange
of molecules
cells and DNA are placed into a cuvette between two
plates.
– High DC voltage (500+ V) applied as a pulse
• square wave form appears to work better than
exponential decay (best for bacteria)
• possible optimizations are voltage, pulse length,
wave form.
– Some experimentation with RF (radio frequency)
pulses suggests greater efficiency
• but apparatus is not readily available
advantages
– very efficient when it works
– quite effective at making stable transfectants (e.g. ES
cells)
disadvantages
– only works well for cells in suspension
• devices for transfecting adherent cells do not
work very well and are cumbersome to clean
– kills cells very effectively
– expensive equipment and cuvettes
– extensive optimization
– very sensitive to salt concentrations
BioSci 145A lecture 11
page 10
©copyright
Bruce Blumberg 2000. All rights reserved
Viral infection
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infection is absolutely the highest efficiency method possible
– 100% infection is routine
DNA to be expressed is cloned into a virus that can infect
your favorite cell type - two general types of virus utilized
– retroviruses (RNA viruses), e.g. HIV
• tend to integrate
• can be insertional mutagens!
• Relatively small sized insert
• narrow host range
– large DNA viruses (adenovirus, vaccinia)
• extrachromosomal replication
• tend to have broad host specificity
• tend to be lytic
• large inserts are possible
many viral genes are not required for infective virions
– nonessential genes are removed, thus allowing the virus
to accommodate foreign DNA.
– Most such viruses requires a packaging strain to get
infective virus particles
• primarily for biosafety
field is primarily driven by gene therapy applications
– most current information found in gene therapy literature
– Pfeifer and Verma (2001) Gene Therapy: promises and
problems Ann. Rev. Genomics Hum. Genet. 2, 177-211
BioSci 145A lecture 11
page 11
©copyright
Bruce Blumberg 2000. All rights reserved
Viral infection (contd)
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advantages
– efficiency
– simplicity of infection
disadvantages
– not really feasible to introduce multiple constructs
per cell. Best for introducing a single cloned gene
that is to be expressed highly
– at least P2 containment required for most viruses
• lots of hoops to jump through with institutional
review boards (IRB)
• viral transfer of regulatory genes, or oncogenes
is inherently dangerous and should be carefully
monitored
• not so many old virologists
– host range specificity may not be adequate
– many viruses are lytic
– need for packaging cell lines
BioSci 145A lecture 11
page 12
©copyright
Bruce Blumberg 2000. All rights reserved
How to get DNA into cells - summary
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common feature of nearly all transfection methods is to
form dense DNA complexes of small, uniform size
– 75-100 nm seems best
how the complex is made may not matter much, many
variations are possible (thousands of papers)
– size uniformity of particles is strongly related to
efficiency of transfection
needs to be optimized for the type of cells and
requirements of each experiment
which method is the best one for me?
– What is working in the lab or surrounding labs?
• Troubleshooting is rate limiting step in science
– liposomes and cationic dendrimers generally the best
• fast
• reproducible
• broad applicability
– if cost is a concern, either make your own liposomes
or use calcium phosphate
– electroporation and viral infection have important
utility but restricted applicability
• electroporation is great for cells in suspension
• viral infection is great for a single gene
single cell microinjection is now feasible (Dr. La Morte)
– throughput is low
– uniform delivery ensures reproducibility
BioSci 145A lecture 11
page 13
©copyright
Bruce Blumberg 2000. All rights reserved
How to get DNA into embryos (other than mouse)
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Why would we want to do this anyway?
– Determine function of identified genes
– develop animal models for various diseases
– confer desirable property
Choice of method depends on model system,
developmental stage and outcome desired
– early embryos
• if cells are large then direct microinjection is
possible (e.g. Xenopus and zebrafish)
• otherwise use methods below
– later embryos, cells are too small for direct
microinjection
• biolistic gene transfer
• electrotransformation
• viral infection
• liposome-mediated transfer
transgenic techniques - germline transmission
– must be using an appropriate system
• mouse
• Xenopus
• Drosophila
• zebrafish
• C. elegans
– not yet in chicken, most amphibians
BioSci 145A lecture 11
page 14
©copyright
Bruce Blumberg 2000. All rights reserved
Embryo microinjection
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Simple, direct way to get DNA, RNA or proteins into
embryos
– primary application is embryos with large cells
(xenopus, zebrafish)
• needles used are ~ 1 m diameter
Xenopus microinjection takes two basic forms
– oocyte injection
– embryo injection
oocyte injection
– oocytes are immature eggs, do not divide
– these are dissected from ovaries and can be used for
various experiments
– DNA must be injected into the nucleus (germinal
vesicle)
• transcription is possible
– RNA must be injected into the cytoplasm
• translation is very robust, can continue for long
periods of time (days)
BioSci 145A lecture 11
page 15
©copyright
Bruce Blumberg 2000. All rights reserved
Embryo microinjection (contd)
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oocyte injection (contd)
– applications
• in vivo expression screening
– microinject pools of mRNA generated from
libraries and evaluate function
– various channels, receptors and transporters
identified this way
• protein expression system
• electrophysiology
– advantages
• long term expression of injected materials
• cells do not divide
• transcription is possible
• apparatus is relatively inexpensive
• easy to collect and store oocytes
• unhurried injections
– disadvantages
• cells do not divide
• not a developing system, limited questions
• nuclear and cytoplasmic injections may be
required
– e.g. reporter gene must be put in nucleus,
mRNA into cytoplasm
BioSci 145A lecture 11
page 16
©copyright
Bruce Blumberg 2000. All rights reserved
Embryo microinjection (contd)
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Embryo microinjection
– typically performed from 1-32 cell stage, depending on
effect desired
– embryos divide and develop
• microinjected materials are mosaically distributed
– no transcription of injected DNA before MBT
• zygotic transcription begins at the midblastula
stage
• by then, microinjected DNA is very mosaic
– transgenic approaches
– RNA is well translated but less stable than in oocytes
(24-36 hrs max)
– applications
• misexpression of mRNAs
• injection of mutant mRNAs
• gain of function
• loss-of-function
– mRNAs encoding dominant negative mutants
– neutralizing antibodies
– RNA-I
– Morpholino antisense oligonucleotides
• Can target injected materials to particular tissues
by using fate maps and blastomere injections at 32
cell stage
BioSci 145A lecture 11
page 17
©copyright
Bruce Blumberg 2000. All rights reserved
Embryo microinjection (contd)
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Embryo microinjection (contd)
– advantages
• very early stages can be manipulated
• targeted injections possible
• possible to combine molecular biology with
experimental embryology
– disadvantages
• no early transcription
• mosaic inheritance
• embryos are dividing
– limited time window for injections
BioSci 145A lecture 11
page 18
©copyright
Bruce Blumberg 2000. All rights reserved
Virus-mediated transfer
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Just as with cultured cells, viral vectors may be used to
express transgenes in embryos
– identical viruses are used (retroviruses and
adenovirus)
– similar host range issues
• use of retroviruses may require use of virus-free
eggs (extremely expensive since most chickens
carry one strain or other of RSV)
clone gene of interest into viral vector
– package into virions
– concentrate and determine titer (infections
particles/volume)
– microinject into embryo
applications
– primary application is with chick embryo
– Juan Carlos Izpisúa Belmonte
advantages
– relatively efficient
disadvantages
– no expression in early embryos!
– may be impossible to express some genes
• e.g. DN-RAR
– retroviruses do not stay at site of injection
– survival issues
– non-specific effects
BioSci 145A lecture 11
page 19
©copyright
Bruce Blumberg 2000. All rights reserved
Biolistic gene transfer
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Somewhat bizarre method developed for very difficult
problems (plant cells)
very small particles are coated with DNA
– blasted into target tissue
• gunpowder
• compressed air
advantages
– works in systems that are refractory to other methods
• e.g. plant cells
• regenerating limbs
– not very difficult
disadvantages
– equipment requirement
– not particularly efficient
• only a few % of target cells survive and take up
DNA
– tissues must survive partial vacuum
BioSci 145A lecture 11
page 20
©copyright
Bruce Blumberg 2000. All rights reserved
Electroporation
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Just like cultured cells, tissues and embryos can be
transfected with DNA by electric pulse
typical setup consists of a pair of microelectrodes
(usually needles) in close proximity.
– Maneuver this into close proximity of target, add
DNA and zap
applications
– primary use is with chick embryos
– some use of RF transfection in other embryos but not
widely practiced or accepted
advantages
– can work in very early embryos
– can target small areas relatively well
• unlike virus-mediated transfection, the DNA
only gets into cells near the electrode
disadvantages
– equipment requirement
• electrodes must be custom made
– plenty of “touch” is required
– not so many applications yet
• chick embryo
– potential of contamination with bacteria and molds
BioSci 145A lecture 11
page 21
©copyright
Bruce Blumberg 2000. All rights reserved
Transgenic technology
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Transgenesis is either not possible or not feasible in all
model organisms
– typical model organisms of interest are:
• C. elegans
• Drosophila
• zebrafish
• axolotl
• Xenopus
• chicken
• mouse
– transgenic techniques are well developed in
• C. elegans
• Drosophila
• mouse
– becoming reasonably doable for
• Xenopus
• zebrafish
– not readily possible
• chicken
• axolotl
targeted gene disruption only works in a few organisms
– mouse
– C. elegans
BioSci 145A lecture 11
page 22
©copyright
Bruce Blumberg 2000. All rights reserved
“Standard” transgenesis - mouse
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standard transgenesis
– this involved microinjecting DNA into a fertilized
egg (mouse) or embryo (Drosophila)
• some fraction of embryos undergo integration of
DNA into genome
• some fraction of these transmit the transgene in
the germline
Palmiter et al., 1982. Nature.
300, 611-5.
BioSci 145A lecture 11
page 23
©copyright
Bruce Blumberg 2000. All rights reserved
“Standard” transgenesis (contd)
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Each mouse that harbors a transgene and transmits it in
the germline is a “founder”
– founders must be evaluated before proceeding to
large scale breeding and analysis
• keeping mice is EXPENSIVE ~$1.00/cage/day.
– Multiple females can be caged together
– but males must be kept individually
• downstream analysis is very time consuming,
tedious and expensive
what would we like to know about a founder line?
– How many copies of the transgene are present?
• Prepare DNA from tails, do Southern analysis
and compare with DNA standards
• Transgene copy number varies from 1 to several
hundred
• Level of transgene expression is usually
proportional to the number of copies
– is the transgene expressed? Transgenes are not
equally active at all integration sites.
• Northern or Western analysis
– Western is best but requires an antibody.
» produce an antibody to the protein
» engineer the transgene to express myc,
flag or other common epitope
– Northern is more commonly performed
BioSci 145A lecture 11
page 24
©copyright
Bruce Blumberg 2000. All rights reserved
“Standard” transgenesis (contd)
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what would we like to know about a founder line?
(contd)
– is transgene expression as predicted?
• If the transgene is under the control of a tissuespecific promoter (e.g. its own), is it expressed
in the correct tissue at the correct time in
development?
– Tissue Northern blots
– in situ hybridization
• If the transgene is expressed from a ubiquitous
promoter, is it expressed ubiquitously?
– tissue Northerns
– quantitative RNA blotting
– RT-PCR
– is the transgene transmitted faithfully?
• Multiple tandem copies of the same sequences
could be problematic
• are expression levels similar in progeny of
founders?
– Same is desirable
– could be more or less, or even absent
BioSci 145A lecture 11
page 25
©copyright
Bruce Blumberg 2000. All rights reserved
“Standard” transgenesis (contd)
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Applications
– Transgenesis is a gain of function method
• doesn’t speak to necessity of a gene, unless a
mutation is being rescued
– rescue of a mutation
– promoter analysis
• identify temporal or spatial requirements for
expression
• verify function of suspected enhancer elements
– create models for dominant forms of human diseases
– identify effects of misexpression
• particularly with genes showing temporally or
spatially restricted expression, e.g. Hox genes
advantages of transgenic technology
– analysis is performed in vivo
• best test for gene regulation
– much less difficult than targeted disruption
– relatively high efficiency compared with targeting
disadvantages
– gain of function
– no ability to target integration site
– no control over copy number
– injected DNA must contain all regulatory elements
– can’t study transgenes with dominant lethal phenotypes
BioSci 145A lecture 11
page 26
©copyright
Bruce Blumberg 2000. All rights reserved
Gene targeting
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Targeted disruption of genes is very desirable, wave of the
future
– great to understand function of newly identified genes
from genome projects
• produce a mutation and evaluate the requirements
for your gene of interest
– good to create mouse models for human diseases
• knockout the same gene disrupted in a human and
may be able to understand disease better and
develop efficacious treatments
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excellent recent review is Müller (1999) Mechanisms of
Development 82, 3-21.
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enabling technology is embryonic stem (ES) cells
– these can be cultured but retain the ability to colonize
the germ line
– essential for transmission of engineered mutations
– derived from inner cell mass of blastula stage embryos
– grown on lethally irradiated “feeder” cells which help
to mimic the in vivo condition
• essential for maintaining phenotype of cells
BioSci 145A lecture 11
page 27
©copyright
Bruce Blumberg 2000. All rights reserved
Gene targeting (contd)
How to make ES cells
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ES cells are very touchy in culture
– lose ability to colonize germ line with time
– easily infected by “mysterious microorganisms” that
inhibit ability to colonize germ line
• ko labs maintain separate hoods and incubators
for ES cell work
– overall, ES cells depend critically on the culture
conditions to keep them in an uncommitted,
undifferentiated state that allows colonization of the
germ line.
BioSci 145A lecture 11
page 28
©copyright
Bruce Blumberg 2000. All rights reserved
Gene targeting (contd)
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technique
– isolate genomic clones spanning the gene of interest
from an ES cell library
– construct a restriction map of the locus with
particular emphasis on mapping the exons
– create a targeting construct with large genomic
regions flanking the region to be disrupted
– an essential exon(s) must be disrupted such that no
functional protein is produced from the gene
• this should be carefully tested in cell culture
before mice are made
– it is often useful to design the construct such that a
reporter gene is fused to the coding region of the
protein
• this enables gene expression to be readily
monitored and often provides new information
about the gene’s expression
– dominant selectable marker is inserted within
replacement region
– negative selection marker is located outside the
region targeted to be replaced
– DNA is introduced by electroporation and colonies
resistant to positive selection are selected.
– Integration positive cells are subjected to negative
selection to distinguish homologous recombinants
• homologous recombinants lose this marker
BioSci 145A lecture 11
page 29
©copyright
Bruce Blumberg 2000. All rights reserved
Gene targeting (contd)
Targeting vector
electroporate
recombination
positive selection with
dominant selective
marker
negative selection to
identify homologous
recombinants
BioSci 145A lecture 11
page 30
©copyright
Bruce Blumberg 2000. All rights reserved
Gene targeting (contd)
BioSci 145A lecture 11
page 31
©copyright
Bruce Blumberg 2000. All rights reserved
Gene targeting (contd)
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Technique (contd)
– homologous recombination is verified by Southern
blotting
– factors affecting targeting frequency
• length of homologous regions, more is better.
– 0.5 kb is minimum length for shortest arm
• isogenic DNA (ie, from the ES cells) used for
targeting construct. Polymorphisms appear to
matter
• locus targeted. This may result from differences
in chromatin structure and accessibility
problems and pitfalls
– incomplete knockouts, ie, protein function is not lost
• but such weak alleles may be informative
– alteration of expression of adjacent genes
• region removed may contain regulatory elements
• may remove unintended genes (e.g. on opposite
strand)
– interference from selection cassette
• strong promoters driving these may cause
phenotypes
Applications
– creating loss-of-function alleles
– introducing subtle mutations
– chromosome engineering
BioSci 145A lecture 11
page 32
©copyright
Bruce Blumberg 2000. All rights reserved
Gene targeting (contd)
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Applications (contd)
– marking gene with reporter, enabling whole mount
detection of expression pattern (knock-in)
advantages
– can generate a true loss-of-function alleles
– precise control over integration sites
– prescreening of ES cells for phenotypes possible
– can also “knock in” genes
disadvantages
– not trivial to set up
– may not be possible to study dominant lethal
phenotypes
– non-specific embryonic lethality is common (~50%)
– difficulties related to selection cassette
BioSci 145A lecture 11
page 33
©copyright
Bruce Blumberg 2000. All rights reserved